LC-MS/MS Method for the Determination of 21 Opiates and Opiate Derivatives in Urine J. Jones, S. Westwood, T. Liddicoat, L. Pereira, T. Edge Thermo Fisher Scientific, Manor Park, Runcorn, UK
Overview Purpose: An LC-MS/MS method for the extraction of 21 opiates and opiate derivatives from urine is demonstrated in this poster. Method: Using a cation exchange SPE method, sample preparation is fast and reproducible giving excellent recovery levels for each compound. The analysis is carried out on a polar endcapped C18 column. The method presented provides chromatographic separation of the isobaric metabolites of morphine, morphine-3beta-dglucuronide, morphine-6beta-d-glucuronide and hydromorphone-3beta-d-glucuronide, which exhibit the same MRM transitions. Results: The method shows good accuracy and precision with %RSD less than 6.2% for all compounds. The compound recovery is in the range of 83.2 98.7%, with LOQs ranging from 0.2 10 ng/ml. Introduction Opiates such as morphine and codeine are naturally occurring alkaloids found in the poppy plant. Semisynthetic opioids, such as hydrocodone, hydromorphone, oxycodone and oxymorphone, and fully-synthetic, such as fentanyl, methadone and tramadol, are derivatives of opiates but not classified as opiates themselves. Opiates are most commonly used in pain relief, particularly in palliative care to alleviate severe and chronic pain associated with terminal conditions. However, misuse of opiates for recreational use is also common due to the side effects such as sedation and euphoria. An initial method for the quantification of opiates from biological matrix used GC-MS. This required the conversion of the glucuronide conjugates to the free form via acid or enzyme hydrolysis, followed by post extraction derivatization. This approach was not only expensive and time consuming, but it is also difficult to ensure complete hydrolysis [1]. LC-MSMS, not only eliminates the necessity of derivatization, but the chromatographic separation and sensitivity achieved also allows for the successful quantification of the free and conjugated species. In order to extract opiates from the urine matrix for quantitative studies, an approach that can be taken is Solid Phase Extraction (SPE). The extraction detailed here is carried out using Thermo Scientific TM SOLA TM SCX. SOLA is a revolutionary SPE device, which provides an unparalleled performance in terms of reproducibility, extract cleanliness and sensitivity compared to conventional SPE, phospholipid and protein precipitation products. The US Substance Abuse and Mental Health Services Administration (SAMHSA) have established limits for most of the opiates at 2000 ng/ml [2]. However, raised levels of opiates or opiates derivatives have been reported in samples which were sourced from a subject which had consumed poppy seeds from bakery goods. Therefore, the range has been extended for this screening assay to ensure fewer false negatives. Methods 2 LC-MS/MS Method for the Determination of 21 Opiates and Opiate Derivatives in Urine
fewer false negatives Methods Sample Pretreatment A standard spiking solution of 21opiates was prepared in methanol. A working internal standard solution of 12 deuterated opiates was prepared in methanol. 180 µl of blank urine was added to 800 µl of 1% formic acid in water. For standards and quality control (QC) samples, 10 µl of standard spiking solution was added, for blanks 10 µl of methanol was added. For standards and QCs 10 µl of working internal standard solution was added and for blanks 10 µl of methanol was added. All samples were vortexed for 30 seconds and then centrifuged for 5 minutes at 5000 rpm. Sample Preparation Plate type: SOLA SCX 10 mg / 2 ml Conditioning stage: 500 µl methanol then 500 µl water Application stage: 1000 µl of sample at approximately 1 ml/min Washing stage 1: 500 µl 1% formic acid in water Washing stage 2: 500 µl 1% formic acid in methanol Elution stage: 2 x 250 µl methanol / acetonitrile / triethylamine (45:45:10 v/v) Dry down under nitrogen without heat and reconstitute in 200 µl 0.1% formic acid in water. Mix well. Liquid Chromatography Instrumentation: Thermo Scientific TM Accela TM 600 LC system Column: Thermo Scientific TM Hypersil GOLD TM aq, 3 µm, 100 x 4.6 mm Mobile phase A: Water + 0.1% formic acid Mobile phase B: Methanol + 0.1% formic acid Flow rate: 1 ml/min Column temperature: 30 C Injection volume: 20 µl Injection wash solvent 1: Water Injection wash solvent 2: 45:45:10 (v/v/v) IPA / acetonitrile / acetone Thermo Scientific Poster Note PN20815_e 06/13S 3
TABLE 1. LC gradient conditions. Time (minutes) % A % B 0 100 0 3 85 15 8 0 100 9 0 100 9.1 100 0 15 100 0 Mass Spectrometry Instrumentation: Thermo Scientific TM TSQ Vantage Triple Stage Quadrupole Mass Spectrometer TABLE 2. TSQ Vantage TM conditions. Ionization conditions HESI Polarity Positive Spray voltage (V) 4000 Vaporiser temperature ( C) 450 Sheath gas pressure (Arb) 40 Aux gas pressure (Arb) 25 Capillary temp ( C) 275 Collision pressure (mtorr) 1.5 Scan time (s) 0.005 Q1 (FWHM) 0.7 Q3 (FWHM) 0.7 TABLE 3. Compound transition details. Parent S-Lens Quantifier (m/z) (V) (m/z) Compound 4 LC-MS/MS Method for the Determination of 21 Opiates and Opiate Derivatives in Urine Qualifier (m/z) Collision Energies (V) Quantifier, Qualifier normeperidine 234.1 70 160.1 188.1 16,13 norfentanyl 233.1 69 84.1 177.1 18, 35 meperidine 248.1 95 220.1 174.1 21, 20 O-desmethyltramadol 250.1 62 58.1 56.1 16, 41 cis-tramadol 264.1 63 58.1 56.1 16, 46 EDDP 278.1 100 234.1 249.2 30, 23 norhydrocodone 286.1 102 199.1 171.1 27, 36 morphine 286.1 90 165.1 201.1 39, 25 hydromorphone 286.1 83 185.1 157.1 30, 40 hydrocodone 300.1 109 199.1 171.1 29, 38 codeine 300.1 100 215.1 165.1 24, 41 oxymorphone 302.1 91 284.2 227.1 19, 28 noroxycodone 302.1 86 284.1 227.1 16, 24 methadone 310.1 69 265.2 105.0 14, 27 oxycodone 316.1 100 298.1 241.1 18, 28 fentanyl 337.1 96 188.2 105.1 22, 37 morphine-3beta-dglucuronide 462.1 136 286.1 201.0 28, 42 morphine-6beta-dglucuronide 462.1 148 286.1 201.0 30, 40 hydromorphone-3beta- D-glucuronide 462.1 143 286.1 185.0 28, 47 codeine-6beta-dglucuronide 476.1 157 300.1 282.1 29, 25 oxymorphone-3beta-dglucuronide 478.1 120 284.1 227.1 28, 41 fentanyl D5 342.2 110 188.1 105.0 23, 38 codeine D3 303.1 114 215.1 165.1 25, 43 hydromorphone D3 289.1 114 185.0 157.0 29, 41 methadone D3 313.2 78 268.2 105.0 14, 28 morphine D3 289.1 107 201.1 165.1 25, 40 norhydrocodone D3 289.1 108 202.1 174.1 27, 36 noroxycodone D3 305.1 85 287.1 190.1 16, 24 oxycodone D3 319.1 97 301.2 244.1 18, 28 cis-tramadol D3 268.2 65 58.1 56.1 16, 35 meperidine D4 252.1 90 224.2 178.2 21, 20 hydrocodone D6 306.1 105 202.1 174.1 30, 39 oxymorphone D3 305.1 89 287.2 230.1 19, 29
Compound confirmation by ion ratio was achieved for each analyte over the specified concentration range. The % target ratio of qualifier ion to quantifier ion was attained during method development and set in the quantification method. Results Separation of all the isobaric metabolites was achieved using a C18 polar endcapped column. The polar group provides a controlled interaction mechanism by which polar compounds can be retained and resolved, and enables the use of 100% aqueous mobile phase at the gradient start. The separation of the glucuronide metabolites of morphine (3 and 6 position) and also the hydromorphone were baseline resolved and is demonstrated in Figure 1. Figure 2 shows an example chromatogram obtained at the LOQ for fentanyl, demonstrating the sensitivity of the assay. Opiate standards extracted from urine gave linear calibration curves over the dynamic range as specified for each compound (Table 4). QC samples were run in replicates of six at a concentration of 30, 150 and 300 ng/ml. Accuracy was assessed at each standard and QC level, all data points were within ±15% or ±20% at the LLOQ. Table 4 shows the mean relative error (%) for QCM (n=6). The precision at each of the QC levels for each of the 21 compounds is less than 6.2% RSD. Overspikes were analyzed in triplicate at concentrations of 15, 250 and 600 ng/ml and used to calculate recovery. The overall percentage recovery level was calculated using the average recovery at the three QC levels, and was greater than 83% for all compounds (Table 4). Thermo Scientific Poster Note PN20815_e 06/13S 5
FIGURE 1. Representative chromatogram of morphine- 6beta-D-glucuronide (1), hydromorphone-3beta-dglucuronide (2) and morphine-3beta-d-glucuronide (3) SRM, extracted from urine at 500 ng/ml. FIGURE 2. Representative chromatogram of fentanyl SRM, extracted from urine at 0.2 ng/ml. TABLE 4. Dynamic range, coefficient of determination, accuracy, precision and recovery data for extracted opiate and opiate derivative standards. Compound Linearity Range (ng/ml) Coefficient of Mean determination Relative Error (%) at QCM (n=6) (%RSD) QCM at 150 ng/ml (n=6) % Recovery normeperidine 10-500 0.9947-4.9 4.7 92.1 norfentanyl 10-500 0.9958 12.8 4.8 94.1 meperidine 1-500 0.998 11.8 3.7 94.2 O-desmethyltramadol 10-500 0.9976-2.1 6.2 98.1 cis-tramadol 10-500 0.9985 8.1 4.3 97.6 EDDP 1-500 0.9973 7.7 6.2 87.2 norhydrocodone 10-500 0.9973 10.2 4.1 93.9 morphine 10-500 0.9966 2.7 4.2 97.0 hydromorphone 1-500 0.9985 5.3 4.7 96.8 hydrocodone 1-500 0.9981 9 5.8 96.3 codeine 1-500 0.998 6.3 5.0 96.4 oxymorphone 1-500 0.9989 12.1 3.9 95.7 noroxycodone 1-500 0.9985-5.2 5.1 95.7 methadone 1-500 0.9979 5.2 3.2 94.0 oxycodone 1-500 0.9981 9.0 6.1 98.7 fentanyl 0.2-500 0.9985 6.9 3.9 96.8 morphine-3beta-dglucuronide 10-500 0.998 3.3 4.1 87.5 morphine-6beta-dglucuronide 10-500 0.9922 6.3 3.9 83.2 hydromorphone-3beta- D-glucuronide 10-500 0.9954 7.6 4.2 90.9 codeine-6beta-dglucuronide 10-500 0.996 5.5 4.1 89.9 oxymorphone-3beta-dglucuronide 10-500 0.9957 9.4 4.6 84.2 6 LC-MS/MS Method for the Determination of 21 Opiates and Opiate Derivatives in Urine
-D-glucuronide 10-500 0.9957 9.4 4.6 84.2 Conclusion SOLA SCX SPE plates and a Hypersil GOLD aq HPLC column allows for a simple extraction and rapid quantification of 21 opiates or opiate derivatives from urine using multiple internal standards. A Hypersil GOLD aq HPLC column provided the chromatographic separation of the isobaric metabolites of morphine, morphine-3beta-d-glucuronide, morphine-6beta-d-glucuronide and hydromorphone-3beta- D-glucuronide which exhibit the same MRM transitions. LOQs ranged from 0.2 10 ng/ml for all compounds. Extraction recoveries for each compound was in the range of 83.2 98.7%. The method showed excellent accuracy and precision with %RSD less than 6.2% for all compounds. The ion ratio criteria were achieved at each standard and QC level for each compound. References 1. Wang P., Stone J.A., Chen K.H., Gross S.F., Haller C.A., Wu A.H. Incomplete recovery of prescription opioids in urine using enzymatic hydrolysis of glucuronide metabolites. Journal of Analytical Toxicology 2006; 30:570-575. 2. SAMHSA (2010) Manual for Urine Laboratories, National Laboratory Certification Program, 1 October 2010. U.S Department of Health and Human Services www.thermoscientific.com/dionex 2013 Thermo Fisher Scientific Inc. All rights reserved. ISO is a trademark of the International Standards Organization. All other 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. Thermo Fisher Scientific, Sunnyvale, CA USA is ISO 9001:2008 Certified. Australia +61 3 9757 4486 Austria +43 1 333 50 34 0 Belgium +32 53 73 42 41 Brazil +55 11 3731 5140 China +852 2428 3282 Denmark +45 70 23 62 60 France +33 1 60 92 48 00 Germany +49 6126 991 0 India +91 22 6742 9494 Italy +39 02 51 62 1267 Japan +81 6 6885 1213 Korea +82 2 3420 8600 Netherlands +31 76 579 55 55 Singapore +65 6289 1190 Sweden +46 8 473 3380 Switzerland +41 62 205 9966 Taiwan +886 2 8751 6655 UK/Ireland +44 1442 233555 USA and Canada +847 295 7500 PN20815_E 06/13S