Development Of A Fast, Green Lc-ms Method For The Quantification Of Oxycodone, Oxymorphone And Noroxycodone Using Innovative Hplc Column Technology

V. Cooper, L. Payne, T. Fleischmann
BASi®, McMinnville, OR

Purpose

Oxycodone is an opioid analgesic widely used to treat moderate to severe pain. Oxymorphone, a minor metabolite, is itself a potent opioid, and noroxycodone, a major metabolite, may be a source of active metabolites. Simultaneous quantification of oxycodone and these metabolites is desirable to account for differences in individual metabolism of the drug. BASi® developed and validated to FDA standards a sensitive LC-MS/MS method for the quantification of oxycodone, oxymorphone and noroxycodone which has been used to analyze clinical samples. The analytes are well separated on a typical C18, 5 micron HPLC column, but the cycle time of 9 minutes is a hindrance to maximizing throughput. BASi® has sought to improve cycle time while also improving resolution by converting the method to utilize an ultra small particle size C18 column with a conventional HPLC.

Methods

The range of the validated assay is 0.1-100 ng for oxycodone and oxymorphone, and 0.5-100 ng/mL for noroxycodone. Detection is by positive TurboIonspray on a Sciex API-4000. The HPLC column is a 2.1 X 50 mm, 5 micron, Waters XBridge C18. Typical retention times of the analytes are oxycodone 5.2 minutes, oxymorphone 3.5 minutes, and noroxycodone 2.4 minutes. The cycle time is approximately 9 minutes.

For the new, faster method, the chromatography has been altered to accommodate the small particle column [2 x 30 mm, 1.5 micron, Grace VisonHT C18] but the extraction and mass spectrometer conditions remain the same. The analytes are eluted on a steep gradient from 10 to 100% organic with a 1 minute hold at 5 minutes and a flow rate of 0.2 mL/min.


Structures of Oxycodone, Oxymorphone and Noroxycodone

Original Method Conditions

Calibration range: 0.1-100 ng/mL oxycodone
0.1-100 ng/mL oxymorphone
0.5-100 ng/mL noroxycodone
HPLC column: XBridge C18 2.1 x 50 mm, 5µm
Detection: Sciex API-4000 positive TurboIonspray
Retention times: oxycodone ~ 5.2 minutes
oxymorphone ~ 3.5 minutes
noroxycodone ~ 2.4 minutes
Flow rate: 0.400 mL minute
HPLC program: 20 to 60% MPB in 6 minutes, 1 minute
column wash and 2 minute equilibration
Cycle time: approximately 9 minutes

Fast and Green Method Conditions

Calibration range: 0.1-100 ng/mL oxycodone
0.1-100 ng/mL oxymorphone
0.5-100 ng/mL noroxycodone
HPLC column: Vision C18 HL 2 x 30 mm, 1.5µm
Detection: Sciex API-4000 positive TurboIonspray
Retention times: Oxycodone ~ 2.5 minutes
Oxymorphone ~ 1.3 minutes
Noroxycodone ~ 2.6 minutes
Flow rate: 0.200 mL minute
HPLC program: 10 to 100% MPB in 5 minutes,
2 minute equilibration
Cycle time: approximately 7 minutes

Example Chromatography at the LLOQ from Original Method

Example Chromatography at the LLOQ from Fast Method

Comparison of Test Results from ‘Fast Method’ to Original Validation

Oxycodone
0.300 ng/mL
10.0 ng/mL
75.0 ng/mL
Original
Fast
Original
Fast
Original
Fast
Mean 0.325 0.321 10.5 10.2 78.2 76.3
S.D. 0.0258 0.0123 0.544 0.363 5.4 2.16
%CV 7.9 3.8 5.2 3.5 6.9 2.8
%Bias 8.3 6.8 5 2.4 4.3 1.7
n 24 6 24 6 24 6
Oxymorphone
0.300 ng/mL
10.0 ng/mL
75.0 ng/mL
Original
Fast
Original
Fast
Original
Fast
Mean 0.301 0.311 10.6 9.86 78.6 76
S.D. 0.0139 0.0258 0.497 0.305 7.98 1.97
%CV 4.6 8.3 4.7 3.1 10.2 2.6
%Bias 0.3 3.9 6 2.4 4.8 1.3
n 24 6 24 6 22 6
Noroxycodone
1.00 ng/mL
10.0 ng/mL
75.0 ng/mL
Original
Fast
Original
Fast
Original
Fast
Mean 1.06 0.972 9.78 9.87 77.9 74.9
S.D. 0.0832 0.0376 0.445 0.278 5.06 1.11
%CV 7.8 3.9 4.6 2.8 6.5 1.5
%Bias 6 -2.8 -2.2 -1.3 3.9 -0.2
n 24 6 24 6 24 6

Example Standard Curves from Fast and Green Method

Oxycodone

Oxymorphone

Noroxycodone

Advantages of Fast, Green Column

Method Parameter
Original
Fast
Flow rate (mL/min)  0.4 0.2
Cycle time (min)  9 7
Solvent volume per cycle  3.6 1.4
Injections per 24 hours  160 205
Solvent Savings per Injection (mL) 2.2
Solvent Savings over 24 Hours (mL) 288

Conclusion

Preliminary data indicates that the Fast, Green method performs as well as or better than the traditional method. Other considerations for converting to the Green method:

  • ~ 61% savings of mobile phase
  • ~ 28% increase in throughput
  • Higher priced column off sets some of solvent savings
  • Unknown column life, higher operating pressure
  • Further work required to evaluate the reproducibility and ruggedness