Karsten Liegmann (Presenter)
California State Polytechnic University, Pomona
Authorship: Karsten Liegmann (1), David Hall (1), John Laycock, Ph.D.(1), Phil Dimson (1)
1. SPEware Corp., 14180 Live Oak Ave, Suite I, Baldwin Park CA. 91706
| Short Abstract Methods that quantify a wide variety of drug chemistries from a single analysis are increasingly being implemented. An impediment to implementation of these methods is inclusion of gabapentin and pregabalin to these comprehensive methods, as specimens routinely have concentrations in the hundreds of microgram per milliliter range. The concentration and types of organic solvents used in the elution step can be modified to predictably and reproducibly reduce the recovery of gabapentin and pregabalin, while not affecting the complete recovery of analytes requiring high levels of sensitivity. |
Long Abstract
Gabapentin (1-(aminomethyl)cyclohexane acetic acid) and pregabalin (β-isobutyl-γ-Aminobutyric acid) are used primarily to treat seizures and neuropathic pain. Both are recommended as a first line agent for the treatment of pain associated with diabetic neuropathy, post-herpetic neuralgia, and central neuropathic pain by the European Federation of Neurological Societies.
As demand for therapeutic drug monitoring rises, methods that can be used to quantify a wide variety of drug chemistries from a single analysis are increasingly being implemented as they reduce costs and streamline workflow.
An impediment to the easy implementation of these methods is the inclusion of gabapentin (and to a somewhat lesser extent pregabalin) to these comprehensive methods, as urine specimens routinely have concentrations in the hundreds of microgram per milliliter range, with some samples having concentrations a hundred fold greater. The resulting off-scale chromatographic peaks, complicate automatic detection of these analytes, result in potential carry-over, and can be the cause of ion suppression in chromatographic neighboring analytes.
In addition to the other benefits associated with of solid phase extraction; the concentration and types of organic solvents used in the elution step can be modified to predictably and reproducibly reduce the recovery of gabapentin and pregabalin, while not affecting the complete recovery of analytes requiring high levels of sensitivity (buprenorphine, norbuprenorphine, fentanyl, sufentanil, etc.). Using various combinations of ethyl acetate, dichlormethane, 2-propanol, and methanol; the recovery of gabapentin and pregabalin could be adjusted from 10% to complete recovery.
The procedure is as follows: 150 µL of Master Mix (containing 120 µL of 100mM sodium acetate pH 4.8, 20 µL of internal standard mix, and 10 µL of β-glucuronidase solution (Kura BG100 at 100,000 units/mL) were added to SPEware Narrow Bore PSCX 2.5mg 96-well plates, followed by 50 µL of blanks, standards, controls and specimens. The hydrolysis reaction was allowed to proceed for 1 hour at 68oC. The samples were loaded onto the sorbent at flow rates of 1-2 mL/min, the columns were then subsequently washed with 500 µL of deionized water, 200 µL of 100mM hydrochloric acid, and 300 µL of water:methanol 75:25 (v:v). The columns were then dried under nitrogen for 5 minutes and eluted with a variety of elution buffers.
Examples:
1. 80:18:2 dichloromethane:2-propanol:ammonium hydroxide yielded approximately a 15% recovery for gabapentin and pregabalin with high recoveries for most other analytes, with clear and colorless extracts.
2. 68:30:2 ethyl acetate:methanol:ammonium hydroxide yielded approximately a 95% recovery for gabapentin and pregabalin with high recoveries for all analytes, with clear and colorless extracts.
Analyses of the samples were performed on an AB Sciex 5000 LCMS system coupled with a Shimadzu LC-20AD HPLC utilizing a Restek Raptor Biphenyl column. The LC-MS/MS method quantifies 43 therapeutic drugs and metabolites in urine from several drug classes including opiates, benzodiazepines, tricyclic antidepressants, and anticonvulsants.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | SpeWare Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |