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Method Validation of a Drug Panel in Urine by LC-MS/MS

Chia-Ni Lin, 1 JoEtta M. Juenke, 2 and Kamisha L. Johnson –Davis1,2.

1Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT USA, 2ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT USA

Introduction Imprecision Method Comparison

Tricyclic (TCA) can cause serious cardiac complications and Imprecision was determined by analyzing three levels of control material To evaluate accuracy, drug-free urine was pooled and used as a matrix to spike 22 samples with TCA at can be lethal if misused at high doses; as a result, TCA should be monitored. To prepared in quintuplicate at five concentrations for five days (n = 25 for each different concentrations. An additional 33 patient samples were collected and sent to an alternate monitor patient compliance to therapy, urine specimens may be preferred since concentration). laboratory for split sample comparison by gas chromatography-mass spectrometry (GC/MS). Out of a total the collection is non-invasive and the specimen can provide a longer detection of 55 results, there were three false negative results for and two false negative results for window. TCA are frequently monitored by immunoassay; however false Expected Mean Within Run Between Day Total , when compared to compared GC/MS method. The false negative results could be due to the positives and cross reactivity to other cyclic compounds may compromise Drug Sample N (ng/mL) (ng/mL) SD CV (%) SD CV (%) SD CV (%) different sample preparations and positive cut-off concentrations between the two laboratories. No false results. 1 25 100 80.88 4.89 6.05 2.12 2.62 5.33 6.59 2 25 300 270.5 15.68 5.69 10.54 3.83 18.90 6.86 positive results were observed. Amitriptyline 3 25 1000 1029 50.75 4.93 40.28 3.91 64.79 6.30 Linear regression for each is found below. Objective Linearity correlation data 4 25 2500 2567 108.65 4.23 116.02 4.52 158.95 6.19 Analytical Drug Range The purpose for this study was to develop a confirmation method by liquid 5 25 5000 5160.7 200.01 3.88 579.48 11.23 613.02 11.88 1 25 100 76.15 4.12 5.41 2.91 3.82 5.04 6.62 Amitriptyline 100-3000 Slope =1.06 r: 0.9974 y intercept= -23.58 n= 10 Amitriptyline chromatography-tandem mass spectrometry (LC-MS/MS) to determine TCA in 2 25 300 268.25 15.84 5.90 11.83 4.41 19.77 7.37 urine specimens. This LC-MS/MS method can quantify amitriptyline, Nortriptyline 3 25 1000 985.09 57.14 5.80 45.34 4.60 72.94 7.40 Nortriptyline Nortriptyline 110-5000 Slope =0.99 r: 0.9810 y intercept= -141.43 n= 14 4 25 2500 2513.7 100.37 3.99 114.35 4.55 152.15 6.05 nortriptyline, , norldoxepin, , , clomipramine, 5 25 5000 5083.3 193.11 3.80 401.53 7.90 445.56 8.77 Imipramine 300-3000 Slope = 0.90 r: 0.9993 y intercept= 49.24 n= 5 1 25 100 79.24 3.50 4.42 5.00 6.31 6.10 7.70 norclomipramine and . Desipramine 300-3000 Slope =1.19 r: 0.9999 y intercept= -39.49 n= 5 2 25 300 273.72 10.75 3.93 9.16 3.35 14.13 5.16 Imipramine 3 25 1000 975.27 Doxepin 34.48 3.54 39.06 4.01 52.10 5.34 Doxepin 300-3000 Slope =0.99 r: 0.9972 y intercept= -7.57 n= 6 4 25 2500 2571.2 83.81 3.26 105.27 4.09 134.56 5.23 Materials and Method Nordoxepin 5 25 5000 5157.5 211.69 4.10 339.50 6.58 400.09 7.76 Nordoxepin 200-2500 Slope =0.98 r: 0.9995 y intercept= -15.46 n= 6 1 25 100 78.44 5.45 6.95 6.60 8.41 8.56 10.91 Impramine Standards were obtained by Cerilliant and prepared in drug-free urine. The 2 25 300 266.98 11.26 4.22 8.76 3.28 14.27 5.34 Clomipramine 400-2500 Slope =1.20 r: 0.9998 y intercept= -71.34 n= 6 Desipramine internal standard which includes: nortriptyline-D3, doxepin-D3, desipramine-D3, Desipramine 3 25 1000 959.12 39.40 4.11 12.84 1.34 41.44 4.32

Drug analyte concentration by LC-MS/MS (ng/mL) LC-MS/MS by concentration analyte Drug Norclomipramine 200-2500 Slope =0.96 r: 0.9996 y intercept= 9.83 n= 6 imipramine-D3, clomipramine-D3, protriptyline-D3 were added to the patient 4 25 2500 2456.3 76.37 3.11 87.75 3.57 116.33 4.74 Protriptyline 5 25 5000 4971.6 157.51 3.17 241.00 4.85 287.91 5.79 Drug analyte concentration by GC/MS (ng/mL Protriptyline 300-2500 Slope =1.28 r: 1.0000 y intercept= -96.32 n= 4 urine, standards, and controls in microcentrifuge tubes. Protein precipitation was 1 25 100 82.4 4.54 5.51 2.71 3.29 5.29 6.42 ) performed with a methanol/acetonitrile solution, then samples were centrifuged 2 25 300 281.44 17.97 6.38 2.79 0.99 18.18 6.46 Six patient urine samples with positive TCA results were tested by the NexScreen urine cup (NexScreen, Doxepin 3 25 1000 982.36 37.14 3.78 17.61 1.79 41.10 4.18 to pellet the proteins. The supernatant was then injected into a LC-MS/MS 4 25 2500 2509.7 87.15 3.47 67.26 2.68 110.09 4.39 Amedica Biotech Inc.). The TCA results were confirmed by our LC-MS/MS method. The other drugs that (Waters Acquity TQD). Concentrations of TCA were determined by ultra high 5 25 5000 5051.7 216.90 4.29 319.41 6.32 386.09 7.64 were positive by the cup were not confirmed. The TCA confirmation results are listed below. 1 25 100 83.34 5.06 6.08 3.84 4.61 6.35 7.62 performance liquid chromatography with tandem mass spectrometry detection 2 25 300 283.22 12.75 4.50 9.04 3.19 15.63 5.52 (Waters Acquity UPLC TQD, [UPLC-MS/MS]). The instrument was operated in Nordoxepin 3 25 1000 996.76 41.55 4.17 21.24 2.13 46.66 4.68 NexScreen Urine Cup LC-MS/MS multiple reaction monitoring (MRM mode with an ESI source in positive 4 25 2500 2589.8 108.54 4.19 72.76 2.81 130.67 5.05 5 25 5000 5301.3 160.10 3.02 251.54 4.75 298.17 5.62 ionization mode. The method was validated for accuracy, recovery, imprecision, 1 25 100 57.7 5.03 8.71 9.12 15.81 10.42 18.05 linearity, analytical measurement range (AMR), specificity, and carryover. 2 25 300 252.19 12.87 5.10 4.58 1.82 13.67 5.42 THC Coc Opi 300 AMP MET PCP MDMA BAR BZO MTD TCA OXY Drug (ng/mL) Clomipramine 3 25 1000 968.44 28.78 2.97 40.96 4.23 50.06 5.17 Accuracy (% recovery) and imprecision (within-run, between run and total %CV) 4 25 2500 2577.7 75.67 2.94 171.79 6.66 187.72 7.28 GC3 - - + - - - - - + - + - GC3 Doxepin (338), Nordoxepin (649) were within +/- 15% of the target concentrations across the analytical 5 25 5000 5347.1 157.93 2.95 254.31 4.76 299.36 5.60 measurement range. Patient specimens were analyzed and the results were 1 25 100 61.67 4.08 6.61 12.29 19.94 12.95 21.00 2 25 300 235.9 11.33 4.80 7.13 3.02 13.39 5.67 GC4 + - + - + + - - + + + + GC4 Amitriptyline (320) Nortriptyline (629) compared to an alternate laboratory. The concentration of analyte(s) were Norclomipramine 3 25 1000 898.62 28.68 3.19 32.34 3.60 43.22 4.81 calculated from the calibration curve and ion ratios between the analyte(s) and 4 25 2500 2480.3 73.08 2.95 190.11 7.67 203.67 8.21 5 25 5000 5224.8 149.67 2.86 361.69 6.92 391.44 7.49 GC14 - - + - - - - - + - + - GC14 Clomipramine (57) Norclomipramine (366) the internal standards. 1 25 100 84.34 4.52 5.36 7.30 8.66 8.59 10.18 2 25 300 284.5 18.25 6.42 5.80 2.04 19.15 6.73 GC15 - - + - - - - - + - + + GC15 Amitriptyline (219) Nortriptyline (405) Linearity Protriptyline 3 25 1000 1018.2 32.78 3.22 39.18 3.85 51.08 5.02 4 25 2500 2602.9 83.16 3.19 112.78 4.33 140.13 5.38 Linearity was evaluated by analyzing five batches of samples spiked at 5 5 25 5000 5406.6 144.69 2.68 229.41 4.24 271.23 5.02 GC16+- + - ++ - - + +++GC16 Doxepin (331), Nordoxepin (673) concentrations in duplicate over the stated analytical range of the assay. Results are shown below. GC8 - + - - - + - - + - + - GC8 Amitriptyline (7607) Nortriptyline (4554) Linearity correlation data Ion Suppression and Interfering Drug Analytical Range Conclusions Amitriptyline 100-2000 ng/mL Slope =0.99 r: 0.9958 y intercept= 4.98 n= 10 Nortriptyline 100-2000 ng/mL Slope =1.00 r: 0.9966 y intercept= 2.82 n= 10 Substances The calibration curves, spiked in human urine were linear with a correlation coefficient of over 0.99 in the Ion suppression was evaluated by analyzing extracted samples while range of 100– 2000 ng/mL. The coefficient variation for inter- and intra-day precision was within 25% for Imipramine 100-2000 ng/mL Slope = 0.99 r: 0.9988 y intercept= 6.33 n= 10 infusing the compounds of interest. No significant suppression was noted. each analyte at five different concentrations: 100, 300, 1000, 2500, and 5000 ng/mL. There was no Structurally similar compounds and other drugs that may be co-administered Desipramine 100-2000 ng/mL Slope =0.99 r: 0.9983 y intercept= 4.33 n= 10 carryover observed in this assay at the concentration of 5000 ng/mL. Urine TCA testing can be a useful drugs were analyzed and no interferences were found. The table below lists approach in identifying patterns of compliance and misuse. This method will provide confirmation of TCA Doxepin 100-2000 ng/mL Slope =0.99 r: 0.9970 y intercept= 3.80 n= 10 the drugs that were evaluated for interference. in urine by LC-MS/MS. Nordoxepin 100-2000 ng/mL Slope =1.00 r: 0.9969 y intercept= -3.64 n= 10 norsertraline norclozapine Acknowledgements Clomipramine 200-4000 ng/mL Slope = 0.99 r: 0.9989 y intercept= 10.78 n= 10 thioradiazine morphine codeine hydrocodone hydromophone Norclomipramine 200-4000 ng/mL Slope =1.01 r: 0.9979 y intercept= -17.97 n= 10 ARUP Clinical Toxicology Department provided use of their instrumentation. oxycodone oxymorphone dihydrocodone The project was supported by the ARUP Institute of Clinical and Experimental Pathology. Protriptyline 100-2000 ng/mL Slope =1.00 r: 0.9974 y intercept= -2.91 n= 10 Carbamazepine epoxide