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
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Oxycodone
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0.300 ng/mL
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10.0 ng/mL
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75.0 ng/mL
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Original
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Fast
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Original
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Fast
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Original
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Fast
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| 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 |
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Oxymorphone
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|
0.300 ng/mL
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10.0 ng/mL
|
75.0 ng/mL
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|
Original
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Fast
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Original
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Fast
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Original
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Fast
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| 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 |
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Noroxycodone
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1.00 ng/mL
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10.0 ng/mL
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75.0 ng/mL
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Original
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Fast
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Original
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Fast
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Original
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Fast
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| 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
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Method Parameter
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Original
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Fast
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| 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
