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Development of a high-throughput LC-MS/MS assay for pain management panel from urine Hui Qiao, Changtong Hao, Sha Joshua Ye, Lisa Cousins Ionics Mass Spectrometry Group, 1-32 Nixon RD, Bolton, ON Canada L7E 1W2 Introduction Pain is a complex phenomenon. Opiate medications are one of the most popular treatments used by physicians to provide pain relief. Since these medications can induce euphoria as well as pain relief, they are frequently abused by pain patients. These medicines also have side effects including sedation, dizziness, nausea, vomiting, and constipation. All these require that the pain physicians be particularly attentive to their prescribing practices and determine whether patients are compliant with their medication regime. Urine drug testing is commonly used to give confidence to both the physician and the patient that the patient is following the medication regimen and is getting the most benefit from their treatment. As the increase of average age of healthcare patients, and the continuously increasing number and availability of pain management drugs, it has created extremely high demanding for less expensive, faster, more accurate, and reliable solutions for analyzing the presence and concentration of the pain management drugs. The recent adaption of LC-MS/MS technology for pain management drug monitoring has been successful due to its high sensitivity, excellent selectivity, and low detection limit. In the work present here, an LC-MS/MS method for the analysis of a pain management panel comprising 12 analytes has been developed on an Ionics 3Q Series 120 triple quadrupole mass spectrometer. Combined with SLE for urine sample cleanup, this LCMS/MS method provides a faster, more accurate and reproducible solution for the analysis of pain management drugs. The preliminary results show that the LLOQs for all the 12 drugs in neat solution are in the range of 20 to 500 pg/mL with CVs less than 15% over the entire concentration range. The accuracy for all the analytes is about 90-105%. This method also displayed good linearity for all analytes with R2 >0.99. The results of drugs in urine matrix will be completed in the further experiments. Experimental Materials Drug standards were purchased from Cerilliant Corporation. An intermediate analyte mixture stock solution was prepared by combing the analyte stock solutions with H2O:Methanol (80:20, V/V) with 0.1% formic acid at appropriate concentrations. βGlucuronidase was purchased from Sigma-Aldrich. Fresh urine was obtained from healthy male volunteer. All other reagents are solvents used were HPLC grade. Instruments An Ionics 3Q Series 120 Triple quadrupole mass spectrometer system was used for all the drug analysis. This instrument is equipped with heated coaxial flow ion source and HSID (hot source induced desolvation) interface, which offers high ionization efficiency and excellent sample desolvation. This greatly improves the instrument sensitivity and lowers the background noise. A Shimadzu Prominence LC system with Restek 5 um, Ultra II Biphenyl Column, 50x2.1mm at 40°C with a gradient of eluent A (0.1% formic acid in water) and elute B (0.1% formic acid in methanol) was used at a flow rate of 600 uL/min. The total LC gradient cycle is 5 minutes. The injection volume is 10 uL. Urine sample was first hydrolyzed to convert the glucuronide metabolites to native form using β-Glucuronidase. This was done by adding 950 uL ammonium acetate (100mM) pH=5 and 25 uL β-Glucuronidase (5000 units) to 1mL urine sample spiked with drugs. Samples were then incubated for 2 hours at 60°C. Then adding aqueous ammonium hydroxide (2%) about 100 uL to the incubated sample to get a final pH=9. After the sample pretreatment, loading 200 uL pretreated solution to SLE plate for cleanup and applying a short pulse through the pressure manifold to facilitate the flow of sample in the SLE plate. The samples were then eluted with ethyl acetate (1mL) and were dried using vacuum workstation. The sample were then reconstituted with H2O:Methanol (80:20, V/V) with 0.1% formic acid. Results Using this LC-MS/MS method, all the analytes are first examined in the neat solution. They all can be eluted from the column within about 2 minutes with good peak shape and resolution. The overlaid MRM ion traces for the quantifier MRM transitions of all 12 pain management drugs is shown in Figure 1. Fig.1. Overlaid MRM ion traces for all the 12 pain management drugs. The concentration is 5 pg/uL for all the analytes. Figure 2 shows the comparison of the extraction of chromatograph (EIC) at blank and LLOQ for morphine at MRM of m/z at 286.1-165.1 with neat solution. The LLOQ is about 0.448 pg/uL with CV < 11% and accuracy about 90-105%. In the calibration curve, regression is performed using weighting of 1/x, R2=0.997. The urine sample spiked with drugs will be processed following the sample preparation procedure mentioned above. And the results will be completed in the further experiment. Fig.2. Extraction ion chromatograph (EIC) of Morphine with MRM at m/z 286.1-165.1 at blank and LLOQ.