UHPLC/MS: An Efficient Tool for Determination of Illicit Drugs

Transcription

1 Application Note: 439 UHPLC/MS: An Efficient Tool for Determination of Illicit Drugs Guifeng Jiang, Thermo Fisher Scientific, San Jose, CA, USA Key Words Accela UHPLC System MSQ Plus MS Detector Drugs of Abuse Hypersil GOLD PFP Columns Sensitivity Goal Optimize a UHPLC/MS method with respect to stationary phase, mobile phase, and detector settings to achieve picogram level quantitation of fourteen drugs and metabolites employing a 12 minutes separation. Introduction Gas chromatography-mass spectrometry (GC-MS) is commonly employed for the separation and identification of drugs and metabolites in forensic toxicology, using electron impact (EI) or chemical ionization (CI). 1 This methodology has become a gold standard in terms of admissibility and defensibility in court because of its good sensitivity, excellent selectivity and a high degree of standardization. 2 However, laborious and time consuming derivatization procedures and sample clean ups are mandatory in most cases. LC/MS methods eliminate the need to derivatize and often simplify sample preparation. However, long run times and low separation efficiency limit the utility of conventional HPLC. Ultra high performance liquid chromatography (UHPLC) performs separations 5 to 10 times faster than conventional HPLC by employing sub-2 µm diameter particles. The 1-2 second peak widths and relatively high separation efficiency of UHPLC are more competitive with capillary GC, making UHPLC-MS an attractive alternative method for illicit drug analysis. This application note illustrates the separation and detection of a mixture of 14 illicit drugs/metabolites by ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS). The drugs/metabolites are separated on a Hypersil GOLD PFP, 1.9 µm, 100 x 2.1 mm column and detected by a fast scanning single quadrupole mass spectrometer. Experimental Conditions 1. Drug Standard Preparation Pseudoephedrine, ephedrine, amphetamine, methamphetamine, 3,4-methylenedioxy-N-methamphetamine (3,4-MDMA), oxycodone, hydrocodone, clonazepam, noscapine, cocaine, caffeine, tetrahydrocannabinol (THC), cannabinol and cannabidiol standards (1 mg/ml in methanol) were purchased from Alltech-Applied Science (State College, PA, USA). The above fourteen compounds were mixed with the optimized molar ratio in the range of 1 to 100 and diluted to 0.1 ppm with methanol to make the drug mixture standards. 2. Chromatographic Conditions Chromatographic analyses were performed using the Accela UHPLC system (Thermo Scientific, San Jose, CA). The chromatographic conditions were as follows: LC Column: Hypersil GOLD, 1.9 µm, 20 x 2.1 mm Hypersil GOLD, 1.9 µm, 50 x 2.1 mm Hypersil GOLD, 1.9 µm, 100 x 2.1 mm Hypersil GOLD, aq (polar endcapped C18), 1.9 µm, 100 x 2.1 mm Hypersil GOLD PFP (perfluorinated phenyl), 1.9 µm, 100 x 2.1 mm Hypersil GOLD PFP (perfluorinated phenyl), 1.9 µm, 50 x 2.1 mm Column Temperature: 45 C Injection: 1 µl Partial Loop Injection, 25 µl Loop Size Syringe Speed: 8 µl/sec Flush Speed: 100 µl/sec Flush Volume: 400 µl Wash Volume: 100 µl Flush/Wash Source: Bottle with methanol Gradients: Method I Column: Hypersil GOLD PFP 1.9 µm, 100 x 2.1 mm A: Water (0.06% acetic acid) B: Acetonitrile (0.06% acetic acid) C: Methanol (0.06% acetic acid) Time (min) Eluent A% Eluent B% Eluent C% For the other gradient methods used, see Appendix A for details.

2 3. Mass Spectrometer Conditions MS analysis was carried out on a MSQ Plus single quadrupole LC-MS detector (Thermo Scientific, San Jose, CA). The MS conditions were as follows: Ionization: Electrospray (ESI) Polarity: Positive Probe Temperature: 450 C Cone Voltage: 60 V Scan Mode: Full scans ( m/z ) and/or Selected ion monitoring (SIM) ESI Voltage: 4.5 kv Results and Discussion 1. MS Detection Both positive and negative electrospray analysis were performed using the polarity switch function of the Xcalibur software. All of the analytes exhibited higher ionization efficiency in the positive ion mode compared with the negative mode. The MS spectra of the drug standards show both molecular ion signals of [M+H] + and acetonitrile adducts of the form [M+ACN+H] +. For 13 of the analytes, the signal from the molecular ion was more intense than the signal from the acetonitrile adduct. For amphetamine, the most intense signal was from the acetonitrile adduct [M+ACN+H] + at m/z of (data not shown). 2. Separations with Standard Stationary Phases Three columns were evaluated to separate the illicit drug mixtures: Hypersil GOLD, Hypersil GOLD aq and Hypersil GOLD PFP (Figure 1). The UHPLC method with each column type was optimized individually. Hypersil GOLD aq, a polar endcapped C18 phase which offers more retention of polar compounds, did not resolve the early eluting compounds including methamphetamine, oxycodone, caffeine, MDMA and hydrocodone. 3 The separation on Hypersil GOLD aq may have been impaired by interactions between the polar endcapped stationary phase and the polar analytes. Hypersil GOLD, with LI or C18 selectivity, showed improved selectivity for all analytes except caffeine (peak 1) and oxycodone (peak 7). Hypersil GOLD uses highly pure silica and endcapping procedure to minimize unwanted interactions between analytes and the acidic silanols of the silica support. Hypersil GOLD PFP enabled the optimal separation of all 14 analytes by improving the resolution of the earlier eluting compounds. Hypersil GOLD PFP introduces a fluorine group into the stationary phase to improve selectivity towards halogenated compounds, as well as polar compounds containing hydroxyl, carboxyl, nitro or other polar groups Separations using Acetic Acid and Trifluoroacetic Acid (TFA) as Eluent Modifier Trifluoroacetic acid, formic acid and acetic acid can be added into the mobile phase to generate differences in selectivity. Separation of 14 illicit drugs on a Hypersil GOLD PFP column was evaluated by using either trifluoroacetic acid, formic acid or acetic acid as eluent modifier. The separation method with 0.02% TFA (Figure 2A) provided fast separation performance with good resolution and sharp peaks. However, the use of TFA is generally not recommended with MS detection due to its effect on signal suppression. All of the analytes are well resolved with 0.1% formic acid as modifier (Figure 1C), but only when 100% water is used at the beginning of the gradient method (Method C). Prolonged use of 100% water may degrade the stationary phase and shorten the column lifetime, so gradient method C is not suited for routine use. Most of the analytes are well separated with adequate resolution using 0.06% acetic acid as eluent modifier (Figure 2B). However, under such conditions, a few pairs of compounds, such as oxycodone and methamphetamine (peaks 7 & 6), hydrocodone and 3, 4-MDMA (peaks 5 & 8), cocaine and noscapine (peaks 10 & 11), are not baseline resolved. 4. Separations with Hybrid Column Phases Three hybrid stationary phases were evaluated after connecting different stationary phase columns in series: Figure 3A: 50 x 2.1 mm Hypersil GOLD + 50 x 2.1 mm Hypersil GOLD PFP Figure 3B: 50 x 2.1 mm Hypersil GOLD PFP + 50 x 2.1 mm Hypersil GOLD Figure 3C: 100 x 2.1 mm Hypersil GOLD PFP + 20 x 2.1 mm Hypersil GOLD Separations of 14 illicit drugs with these three hybrid stationary phases demonstrated great variation in selectivity. In general, the hybrid column phases improved selectivity between THC and cannabinol, cocaine and noscapine, but reduced selectivity between earlier eluting compounds, such as oxycodone, MA, hydrocodone and MDMA, compared with the Hypersil GOLD PFP phase. 5. Separation with Ternary Gradient The separation of the drug mixtures was dramatically improved by using three solvents: water, acetonitrile and methanol (Figure 4). Baseline resolution of all 14 drugs was achieved. Methanol, a weaker eluent compared with acetonitrile, provided better resolution for most of the analytes. However, the flow rate had to be reduced to accommodate high column backpressure caused by the high viscosity of methanol. Adding acetonitrile reduced the column backpressure so as to maintain the same separation speed.

3 Figure 1: Comparison of 1.9 µm Hypersil GOLD stationary phases for the UHPLC separation of 14 illicit drugs. A) Hypersil GOLD aq, Method A was applied; B) Hypersil GOLD, Method B was applied; C) Hypersil GOLD PFP, Method C was applied. See Appendix A for methods details. 6. Calibration Curve and Sensitivity Calibration curves for the drug standards were constructed over the concentration range listed in Table 1 with 10 calibration levels (Figure 5). Each calibration level was injected three times and the mean area responses were plotted against the concentrations. Correlation coefficients with R 2 = or better were achieved for all illicit drug compounds. The limit of quantitation (LOQ) and the limit of detection (LOD) of the drug compounds were determined based on the calibration curve of signal-to-noise ratio versus concentration and the definitions of LOQ and LOD using s/n = 10 and 3, respectively. LOQs for all drugs were in the range of ng/ml, while LODs were from 0.29 to 90.0 ng/ml (Table 1). The outstanding sensitivity by this method was highlighted by the achievement of picogram level quantitation for 10 illicit drugs with 1 µl sample injection. LOQ LOD Linear Range Analyte (ng/ml) (ng/ml) (ng/ml) ephedrine pseudoephedrine amphetamine methamphetamine ,4-MDMA hydrocodone oxycodone clonazepam cocaine noscapine cannabidiol cannabinol THC Table 1: LOQ and LOD of the thirteen drug compounds with 1 µl sample injection. Figure 2: UHPLC/MS chromatograms of the 14 illicit drugs with acidic solvent modifiers. A) 0.02%TFA (Method D); B) 0.06% acetic acid (Method E). See Appendix A for methods details.

4 Conclusions Fourteen illicit drugs and metabolites are baseline separated in twelve minutes by employing UHPLC/MS with a ternary solvent gradient. Various selectivities are achieved by different column surface chemistry, acidic solvent modifier and eluent system. These results are useful for method developments of drug identification and quantitation. Detection by single quadrupole MS at the ppb (ng/ml) level is more than sufficient to identify and quantify illicit drugs in real samples. References 1. C. Koeppel and J. Tenczer: Scope and limitations of a general unknown screening by gas chromatography-mass spectrometry in acute poisoning. J. Am Soc. Mass Spectrom. 1995, 6, W. Weinmann, A. Wiedemann, B. Eppinger, M. Renz and M. Svoboda: Screening for drugs in serum by electrospray ionization/collisioninduced dissociation and library searching. J. Am Soc Mass Spectrom. 1999, 10, Catalog, Chromatography Columns and Consumables, 08 09, Thermo Scientific, page Figure 3: Comparison of hybrid stationary phase chemistry for the separation of 14 illicit drugs. A) 50 x 2.1 mm Hypersil GOLD + 50 x 2.1 mm Hypersil GOLD PFP, Method F; B) 50 x 2.1 mm Hypersil GOLD PFP + 50 x 2.1 mm Hypersil GOLD, Method G; C) 100 x 2.1 mm Hypersil GOLD PFP + 20 x 2.1 mm Hypersil GOLD, Method H. See Appendix A for method details. Figure 4: Optimized UHPLC/MS separation of 14 illicit drugs with ternary gradient, listed in Method I.

5 Figure 5: Calibration curves for illicit drugs.

6 Appendix A In addition to these offices, Thermo Fisher Method A Method D Method G Scientific maintains Column: Hypersil GOLD aq, 1.9 µm, 100 x 2.1 mm A: Water, 0.1% FA B: Acetonitrile, 0.1% FA Flow Rate: 750 µl/min Method B Column: Hypersil GOLD, 1.9 µm, 100 x 2.1 mm A: Water, 0.1% FA B: Acetonitrile, 0.1% FA Method C Column: Hypersil GOLD PFP, 1.9 µm, 100 x 2.1 mm A: Water, 0.1% FA B: Acetonitrile, 0.1% FA Column: Hypersil GOLD PFP, 1.9 µm, 100 x 2.1mm A: Water, 0.02% TFA B: Acetonitrile, 0.02% TFA Method E Column: Hypersil GOLD PFP, 1.9 µm, 100 x 2.1 mm Method F Hypersil GOLD, 1.9 µm, 50 x 2.1 mm Hypersil GOLD PFP, 1.9 µm, 50 x 2.1 mm Hypersil GOLD PFP, 1.9 µm, 50 x 2.1mm Hypersil GOLD, 1.9 µm, 50 x 2.1 mm Method H Hypersil GOLD PFP, 1.9 µm, 100 x 2.1 mm Hypersil GOLD, 1.9 µm, 20 x 2.1 mm a network of representative organizations throughout the world. Africa Australia Austria Belgium Canada China Denmark Europe-Other France Germany India Italy Japan Latin America Middle East Netherlands South Africa Spain Sweden/Norway/ Finland Switzerland UK USA Legal Notices 2008 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific Inc. products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. View additional Thermo Scientific LC/MS application notes at: Thermo Fisher Scientific, San Jose, CA USA is ISO Certified. AN62904_E 10/08M Part of Thermo Fisher Scientific