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, 5 µm |
| 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, 1.5 µm |
| 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
|
