APPLICATIO OTE Liquid Chromatography/ Mass Spectrometry Authors: Avinash Dalmia Joanne Mather PerkinElmer, Inc. Shelton, CT Workflow for Screening and Quantification of the SAMHSA (IDA) Panel in Urine Using UHPLC-TOF Introduction Workplace drug screening is a common feature of hiring practices as is routine screening of the existing workforce, such as federal employees, safety sensitive employees (e.g., railroad and airline employees) and non-federal non-regulated employees. Transportation employees are the largest group tested. Drug testing is also widely used in continuing probation cases and federally approved release cases, and in medical screening settings for pain medication compliance. In the U.S., drug screening for people working in certain occupations is mandated and regulated by the Substance Abuse and Mental Health Services Administration (SAMHSA).
The SAMHSA panel (formally referred to as the IDA panel) classes are chosen to represent the most commonly abused drugs in the general public. This list includes cocaine, marijuana, amphetamines, opiates and phencyclidines. The full list of compounds with their screening and confirmatory cutoff levels in urine (the most widely used matrix) is displayed in Table 1 1. A list of all compounds with their associated acronyms and molecular structures is displayed in Table 2. Table 1. List of analytes and their cutoff levels. Initial Test Analyte Initial Test Cutoff Concentration Confirmatory Test Analyte Confirmatory Test Cutoff Concentration Marijuana metabolites 50 ng/ml THCA 1 15 ng/ml Cocaine metabolites 150 ng/ml Benzoylecgonine 100 ng/ml Opiate metabolites 2000 ng/ml Codeine 2000 ng/ml Codeine/Morphine 2 Morphine 2000 ng/ml 6-Acetylmorphine 10 ng/ml 6-Acetylmorphine 10 ng/ml Phencyclidine 25 ng/ml Phencyclidine 25 ng/ml Amphetamines 3 500 ng/ml Amphetamine 250 ng/ml AMP/MAMP 4 Methamphetamine 5 250 ng/ml MDMA 6 500 ng/ml MDMA 250 ng/ml MDA 7 250 ng/ml MDEA 8 250 ng/ml 1. Delta-9-tetrahydrocannabinol-9-carboxylic acid (THCA). 2. Morphine is the target analyte for codeine/morphine testing. 3. Either a single initial test kit or multiple initial test kits may be used provided the single test kit detects each target analyte independently at the specified cutoff. 4. Methamphetamine is the target analyte for amphetamine/ methamphetamine testing. 5. To be reported as positive for methamphetamine, a specimen must also contain amphetamine at a concentration equal to or greater than 100 ng/ml. 6. Methylenedioxymethamphetamine (MDMA). 7. Methylenedioxyamphetamine (MDA). 8. Methylenedioxyethylamphetamine (MDEA). Table 2. List of analytes and their molecular structures. Compound Amphetamine (AMP) Methamphetamine(MAMP) 3,4-Methylenedioxyamphetamine(MDA) 3,4-Methylenedioxymethamphetamine(MDMA) 3,4-Methylenedioxy--ethylamphetamine (MDEA) Phencyclidine (PCP) Codeine (COD) Morphine (MOR) 6 Acteylmorphine (6-AM) Benzoylecgonine (BZE) Tetrahydrocannabinol carboxylic acid (THC-COOH) Structure Immunoassays have traditionally been used for screening of the SAMHSA panel but this approach can be challenging since it can give false positive results. Some over the counter medications can produce a false positive result, such as decongestants, yielding a positive result for amphetamine. These false positives then require confirmation by other complimentary techniques, such as GC/MS. Immunoassays are not always sensitive enough to detect low levels of the drug in challenging matrices, such as urine and blood and can often not identify specific drugs within drug classes due to their lack of specificity. An example of this is the detection of morphine where the test would be unable to determine whether the drug taken was morphine, codeine or heroin. GC/MS, used extensively in confirmatory analysis, offers its own challenges in the analysis of drugs of abuse. Most of the compound classes are polar and often thermally labile thus requiring derivatization prior to analysis. Thermally labile compounds are often misidentified due to common EI fragments with other compounds. Unlike GC/MS, LC/MS does not require time consuming derivatization of samples and is ideally suited for the rapid analysis of these compounds. In 2011, SAMSHA altered the guidelines to allow LC/MS instruments to be used for urine quantitative confirmatory analysis. Among the LC techniques, LC/MS/MS is often used to quantitate drugs of abuse in biological fluids due to its sensitivity and selectivity. However, triple quadrupole techniques can have an undesirable high cost and lack the ability to easily identify new or unknown compounds. 2
We present an alternative technique to quantitate drugs of abuse in urine utilizing a rapid dilute and shoot with LC separation method in combination with time-of-flight mass spectrometry (TOF MS). The detection limits of the SAMHSA panel analyzed by the TOF were approximately 1.5-1000 times lower than those required by the SAMHSA guidelines for a confirmatory test cutoff level. In addition to the wide quantitative dynamic range of the AxIO 2 TOF MS, which rivals capabilities of the triple quadrupole instruments, the TOF also provides full spectrum information which allows for screening of non-target compounds. Due to the variety of the illicit and abused drugs available and high incidence of drug abuse, it is vital that labs have an approach that is fast, yet generic in nature and not targeted. It is costly and time prohibitive to add new drugs to an immunoassay based screening panel. TOF MS collects all the ions, and can be used to screen for a new drug immediately at little to no extra method development or cost, and with no requirement to reanalyze the sample. In this application note we present a rapid workflow for the screening and quantification of drugs of abuse in urine. Experimental A workflow for the screening and quantification of the IDA panel is shown in Figure 1. Sample preparation Urine (0.5 ml) was diluted with 0.5 ml of water. Sample (10 µl) was directly injected on column. o sample extraction was required. Calibration Curve(s) Urine blanks were spiked with calibrant levels of 11 IDA panel drugs and 300 ng/ml of deuterated internal standards of 10 IDA panel drugs. The deuterated MDMA standard was used as an internal standard for both MDA and MDMA. Samples were diluted 1:1 with water and 10 µl injected onto column. Each calibration level was injected five times. LC chromatography was developed to ensure that no interferences inherent in the matrix were detected as false positives, and also to ensure minimization of matrix effects (suppression or enhancement). LC conditions: Pump: PerkinElmer Flexar FX-15 UHPLC pump Flow: 0.25 ml/min Mobile phase A: 100 % Water with 10 mm Ammonium Formate adjusted to ph 5.5 Mobile phase B: 95 % AC/5% Water with 0.05 % Formic Acid Gradient conditions: Time (min) %A %B Curve 1.0 97.5 2.5 5.5 25 75 1 8.5 5 95 1 10.0 5 95 1 Injection volume: 10 µl Full Loop Mode Column: PerkinElmer Brownlee SPP column C-18, 2.1x50 mm, 2.7 μm (part number 9308402), SPP C18 guard column cartridge 2.1 mm x 5 mm, 2.7 μm (part number 9308513), guard column holder (part number 9308534) Column temperature: 30 C Diverter valve: LC Effluent was diverted to waste during initial 1.8 min of LC runs conditions: Mass spectrometer: Ionization source: Ionization mode: Acquisition mode: Internal calibration: PerkinElmer AxIO 2 TOF MS PerkinElmer Ultraspray 2 (Dual ESI source) Positive Trap Pulse Performed using m/z 118.0863 and 322.0481 as lock mass ions. Targeted Compounds to Screen Dilute and shoot sample Compound detected /? AALTE THC-COOH COCAIE CODEIE MORPHIE MDA MDEA O ES Report egative Screen Quantification & Confirmation Accurate Mass LC/MS Interrogate Data ID Unknowns 6-AM PCP BZE AMP MAMP MDMA Figure 1. Workflow for screening, identification and quantification of SAMHSA panel in urine. 3
Results Screening To rapidly identify the presence or absence of drugs in large batches of samples, AxIO Solo software was used. AxIO Solo provides quick visualization of the presence or absence of analytes in the samples (Figure 2). Presence of individual drugs can be coded with a specific color for ease of identification. The software identifies the presence of a drug based on accurate mass and isotope profile ratio as shown in Figure 3. The isotope ratios allow further confirmation of the identity of detected compound, lowering the risk of false positives and can also be used to add confidence to the assignment of chemical composition to unknown species. In addition to searching against spectral information, the software also searches for target analytes based on user defined retention time windows which further improves the specificity of detection. The list of target analytes can be quickly and easily added to as previously unknown analytes are detected in samples. The analysis of the SAMHSA panel was completed in < 10 min. (Figure 4) with all peaks eluting before 9.5 minutes. The acquisition rate of the AxIO TOF 2 is sufficient to provide a total of at least 10 spectra across each chromatographic peak, as required by regulations. The use of the divert valve, which removes the salt to waste, ensures a cleaner more robust assay as urine salts, which tend to elute at the beginning of the chromatographic run do not enter the mass spectrometer. Figure 2. AxIO Solo screen shot for blank urine and 100 ng/ml levels of IDA drugs in urine. [M+H] + Theoretical Mass = 208.1332 Measured Mass = 208.1335 Mass Accuracy = 1.44 ppm MDEA M+1 Isotope Expected Rel. Response = 13.05% Measured Rel. Response = 13.43% Figure 3. Mass accuracy and isotope profile of MDEA. MDEA M+1 Isotope Expected Rel. Response = 0.77% Measured Rel. Response = 1.28% Figure 4. EIC for 300 ng/ml of 11 IDA panel drugs standard in urine. Figure 5. MDEA- linear calibration curve (1-1000 ng/ml). 4
Confirmation/Quantification The overall assay sensitivity was determined to be in the 1-10 ng/ ml range for all of the drugs spiked into urine, (Table 3). The limit of quantification (LOQs) measured by the TOF instrument were 1.5-1000 times more sensitive than what is currently required by the SAMHSA guidelines for confirmatory analysis. When analyzing such low levels of compound carryover must be assessed to ensure that the assay is suitable for use. In spite of the low LOQs provided by the TOF MS, 0% carryover was observed for the majority of the analytes and levels were negligible in others were detected, after an injection of the upper limit of quantification (ULOQ) mixture of the drugs tested. Linearity of a representative drug MDEA is shown in Figure 5. The assay showed linearity over three orders with an r 2 value of 0.9995. The majority of the drugs of abuse analyzed showed linearity of three orders of dynamic range where all data was processed without weighting and did not require a quadratic fit, with r 2 values of 0.99 demonstrating that the assay was linear and valid over the clinically relevant range required (Table 4). Multiple injections (n=5) of each calibration level showed excellent reproducibility (RSDs< 15%) for each of the drugs. The presence of a given drug in a urine sample can be confirmed by accurate mass and isotope profile provided by TOF MS. As shown in Table 5, the accurate masses of the majority of the drugs of abuse are < 3 ppm. The cannabinoid metabolite (delta-9-tetrahydrocannabinol-9- carboxylic acid/ THC-COOH), a difficult analyte to ionize, was detected with LOQ of 10 ng/ml which is below the confirmatory cutoff level required. Morphine, a difficult analyte to retain and remove potential interferences simultaneously, was detected with LOQ of 10 ng/ml. Table 3. IDA panel drugs of abuse LOQ in urine with associated cutoff levels. Initial Test Confirmatory LOQ Cut Off Test Cutoff Analyte (ng/ml) Level (ng/ml) Level (ng/ml) AMP 10 500 250 MAMP 3 500 250 MDA 10 500 250 MDMA 3 500 250 MDEA 1 500 250 PCP 1 25 25 BZE 3 150 100 6-AM 10 10 10 Morphine 10 2000 2000 Codeine 3 2000 2000 THC-COOH 10 50 15 Table 4. IDA linearity correlation coefficients. Analyte Concentration Range (ng/ml) r 2 AMP 10-10000 0.9966 MAMP 3-10000 0.9979 MDA 10-10000 0.9982 MDMA 3-10000 0.9968 MDEA 1-1000 0.9995 PCP 1-10000 0.9901 BZE 3-10000 0.9955 6-AM 10-10000 0.9959 Morphine 10-10000 0.9990 Codeine 3-10000 0.9955 THC-COOH 10-10000 0.9944 Table 5. Exact mass and formula for 11 IDA panel drugs of abuse. Compound [M+H] + Formula Measured Mass Mass Error/Da Mass Error/ppm AMP 136.1120 C 9 H 13 136.1114 0.0006 4.4 MAMP 150.1277 C 10 H 15 150.1272 0.0005 3.5 MDA 180.1019 C 10 H 13 O 2 180.1012 0.0007 3.9 MDMA 194.1176 C 11 H 15 O 2 194.1168 0.0008 3.9 MDEA 208.1332 C 12 H 17 O 2 208.1326 0.0006 2.9 PCP 244.2059 C 17 H 25 244.2053 0.0006 2.8 Morphine 286.1438 C 17 H 19 O 3 286.1434 0.0004 1.4 BZE 290.1387 C 16 H 19 O 4 290.1385 0.0002 0.7 Codeine 300.1594 C 18 H 21 O 3 300.1606 0.0012 4.0 6-AM 328.1543 C 19 H 21 O 4 328.1552 0.0009 3.9 THC-COOH 345.2060 C 21 H 28 O 4 345.2068 0.0008 2.2 5
Conclusions The method required little to no sample preparation or method development, saving hours of time and the use of costly reagents and consumables. This equates to a much lower cost per sample. The AxIO 2 TOF was easily able to screen and confirm 1-10 ng/ ml concentrations of drugs of abuse spiked in urine. The detection limits of these drugs were up to 1000 times lower than that required by the SAMHSA guidelines. AxIO 2 TOF with the ADC detector technology provides wide dynamic range capabilities similar to that of a triple quadrupole mass spectrometer, and although not required by the federal regulations, offers the screening of untargeted compounds and allows for subsequent re-interrogation of data. The AxIO 2 TOF MS is much easier to set up and adjust current methods for new or unknown compounds in comparison to triple quadrupoles which are more time consuming when modifying current methods or developing new methods. For rapid large scale screening of batches of samples, PerkinElmer AxIO Solo software provides a quick and easy platform to detect the presence or absence of drugs of abuse. References 1. http://www.workplace.samhsa.gov/drugtesting/pdf/ 2010GuidelinesAnalytesCutoffs.pdf PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA P: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com For a complete listing of our global offices, visit www.perkinelmer.com/contactus Copyright 2013-2014, PerkinElmer, Inc. All rights reserved. PerkinElmer is a registered trademark of PerkinElmer, Inc. All other trademarks are the property of their respective owners. 011498A_01