Simple and Rapid Method Development for High Throughput Analysis of Pain Management Drugs in Urine by LC-MS/MSSha Joshua Ye ; Frenny Ruparelia; George Scott
IONICS Mass Spectrometry Group, Inc, Bolton, Ontario, CANADA

Introduction

Results

LC-MS/MS has emerged as a reliable method to perform both screening and confirmation of the presence ofdrugs (of abuse or for pain management). Routine testing of urine or other biological specimens for prescribed
pain medication is critical to monitor compliance and prevent misuse or abuse. Urine is used for analysis because it can be collected easily. However, patient urine composition is highly variable and dependent their diet,health and lifestyle. These variations in the urine matrix can have an adverse impact on the chromatographic
separation and LC-MS/MS signal. The present study demonstrates that a simple dilute and shoot method
coupled with a highly sensitive LCMS system is feasible to eliminate a typical sample clean-up step withoutcompromising quantitation quality.

LC-MRM Chromatograms of Fentanyl and Norfentanyl in Water

Calibration Curves
Fentanyl: 1 to 10,000 fg/µL

Fentanyl: blank, 250 and 500ag/µL in duplicate injections

Norfentanyl: 1 to 10,000 fg/µL

500ag/µL
250ag/µL
Blank

S/N=13S/N=6Method
Sample and Preparation: The Fentanyl, Norfentanyl, Pentazocine and Meperidine standard stock solutions inliquid form were purchased from Cerilliant Inc (Round Rock, Texas) and stored at -20°C. For sensitivity test, thelow level of pure Fentanyl and Norfentanyl standards were prepared by diluting the high concentration stockwith 50/50 water/methanol in 0.1% formic acid. The drug free urine was purchased from UTAK (Valencia, CA).
The urine was cleaned up by centrifuge for 15 minutes and the supernatant was filtered by 0.2µm filter, then it isdiluted by 0.1% formic acid 1000 times and used as urine matrix. The calibration standards were prepared bymaking an addition of ten microliters of working solution to the diluted urine to obtain required concentrationlevels.
LC-MS/MS Conditions: The LC-MS/MS was performed using IONICS’ 3Q 200 Series,a triple quadrupole mass
spectrometer (Bolton, ON Canada) with a Shimadzu UFLC system. Sample injections of 10µL were loaded on aImtakt Cadenza CD-C18HT column (50x2.0mm, 3µm) using the gradient (60% B isocratic for sensitivity test) asshown below at a flow rate of 0.5mL/min. Mobile phase A: 5% MeOH, 95% H2O, 0.1% Formic Acid,5mM
NH4OAc; Mobile phase B: 95% MeOH,5% H2O, 0.1% Formic Acid, 5mM NH4OAc. The mass spec ion source
settings are: Electrospray Voltage = 5000V, HSID=250°C, Nebulizer Gas=450, Drying Gas=200, Heating
gas=350, Source temperature =325°C. And the optimized MRM parameters for all analytes are shown in thefollowing table.

Norfentanyl: blank, 250 and 500ag/µL in duplicate injections
500ag/µL
250ag/µLBlankPentazocine: 4 to 10,000 fg/µL

S/N=13Meperidine: 2 to 10,000 fg/µL

S/N=6Extracted Ion Chromatograms of Four Mixed Compounds in Diluted Urine

Signal to Noise Ratio (S/N)

Fentanyl (1 fg/µL, retention time = 2.46 min

The calibration curves generated for Fentanyl (377.3/188.1), Norfentanyl (233.3/84.1), Pentazocine(286.3/69.1) and Meperidine(248.3/220.1) with triplet injections using 1/x weighting showed good linearity (R2 > 0.999) up to 4 orders of magnitude in concentrations ranging from low femtograms per microliter to 10,000 fg/µL. The average accuracy and CV% at LLOQ are 99.1% and 15.5% for Fentanyl,
94.5% and 13.0% for Fentanyl, 104.4% and 18.2% for Pentazocine, 106.7% and 11.6% for Meperidine, respectively.

Norfentanyl (1 fg/µL, retention time = 2.24 min)

The matrix is minimized by diluting the urine (a thousand times in present study) and an excellent
specificity is maintained through the LLOQs.

Pentazocine (4 fg/µL, retention time = 2.39 minConclusion
A detection limit in the attogram level is found for Fentanyl and Norfentanyl with IONICS 3Q 200series Molecular Analyzer triple quadrupole mass spectrometer.
Meperidine (2 fg/µL, retention time = 2.34 min)

IONICS Mass Spectrometry

32 Nixon Road, Bolton, Ontario, Canada

A fast, sensitive, and accurate LC-MS/MS method based on “dilute and shoot” methodology for Fentanyl, Norfentanyl, Pentazocine and Meperidine using IONICS 3Q 200 series Molecular Analyzer wasdeveloped. The LLOQs achieved for these compounds in diluted urine were at low femtogram per microliter levels with 10µL injection. Excellent linearity over four orders of magnitude in concentration isalso obtained with good precision and accuracy for these compounds . This method requires little
sample preparation and is well-suited for routine drug of abuse quantitation.

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