Morphine Extraction

[lucyfash]

Hello,

I have been trying to successfully and reproducibly extract morphine and codeine from human plasma. Because we are trying to get 5-100 ng/mL morphine and codeine, each, our UV detection is set low (210) bringing out everything and our fluorometer is set at 220/340. I have increased the column temperature, tested various wavelengths, buffers, and still no reproducible morphine. Codeine is coming out fine. Hydromorphone is our current IS.

Buffer: 1M Carbonate, pH 10
Extraction Solvent: Methylene Chloride
Column: Nucleosil 100 (C18) 5µm, 150mm x 4.6mm
Guard: Nucleosil 100 (C18) 5µm, 7.6mm x 4.6 mm
Pump flow: 1mL/min
Mobile Phase: 0.1M sodium dihydrogen phosphate, pH 3.3, 36% ACN, and 5mM SDS
Sample reconstituted in: water or MP (water has not been giving me a clean baseline)
Injection volume: 50µL
Column temp: 35°C
Degasser: On
Wavelength: 210

I have also tried unsuccessfully a cyano column using ACN:TEA:water (4:0.1:95.9).

Any advice will be appreciated.

Lucy

[Uwe Neue]

It is not clear to me, if your problem is inconsistent retention, or inconsistent extraction results. Please clarify!

[lucyfash]

Sorry, I have been getting inconsistent extraction results. The recovery of morphine has been in the 10% area. Some days are better, some days are worse.

[Uwe Neue]

OK... 10% is not a good starting point for the extraction. I suppose that this is where the problem starts. We need to figure out a way to get a more complete extraction or change the extraction method completely.

What is the extracted fraction for codeine? Is it much higher?

In order to get your analytes quantitatively into the methylene chloride, they should not be in an ionic state. This might be bit tricky to accomplish with the two phenol groups and the tertiary amine on the morphine. I can see why codeine could work better than morphine.

If the extraction of codeine is indeed much higher, this would indicate that this thoughtprocess is on the right track.

[JMB]

Lucy,

A search of "Google Scholar" for "morphine extraction human plasma" gives about 3200 hits, including many refs. to solid phase extraction

e.g. J Chromatogr B Biomed Sci Appl. 2000 May 26;742(1):115-23.

High-performance liquid chromatographic determination of morphine and its 3- and 6-glucuronide metabolites by two-step solid-phase extraction.

Meng QC, Cepeda MS, Kramer T, Zou H, Matoka DJ, Farrar J.

Center for Research in Anesthesia and Anesthesia, Department of Anesthesia, University of Pennsylvania Health System, Philadelphia 19104, USA.

To provide more accurate measurement of morphine and its metabolites for a study of the genetic differences on morphine response, a method for the analysis of morphine and its metabolites is described which has the advantages of increased sensitivity and specificity by using a cleaner extraction. The new extraction method involves both the hydrophobic isolation on a carbon cartridge and ion-exchange isolation on ion-exchange resin which has not preliminary been described for morphine analysis. The combination of these two steps successfully purified drugs from human plasma with maximum removal of interfering substance comparing with a conventional C18 cartridge alone. The analytes are quantified by high-performance liquid chromatography on a reversed-phase C18 column employing a mobile phase consisting of 25% acetonitrile in 0.05 M phosphate buffer (pH 2.1), and 2.5 mM sodium dodecyl sulfate as the pairing ion with a combination of electrochemical and fluorometric detections. The recoveries for morphine (M), morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G) and hydromorphone after the SPE procedure were 86+/-7.1%, 82+/-6.9%, 79+/-6.0% and 85+/-6.0%, respectively. Limits of detection for this method are 0.1 ng/ml for M, and 0.18 ng/ml for M3G and M6G. Limits of quantitation were approximately 0.25 ng/ml for M, and 0.45 ng/ml for M3G and M6G. The present assay was applied to measure M, M3G and M6G content in human plasma to test the applicability and suitability of this method for clinical and research use.

JMB

[Uwe Neue]

May I add the following reference for codeine and its glucuronide:

Yung-Fong Cheng, Uwe D. Neue. Robert Bonin, Eric Block, Laura Bean, "Simplified procedure for the determination of codeine and its metabolite in urine and plasma by LC/UV and LC/MS using mixed-mode cation exchange for sample preparation", J. Liq. Chromatogr. & Rel. Technol. 24(9), 1353-1364 (2001)

This paper also includes data for other drugs out of human plasma using essentially the same technique.

And, of course, you have the author here to give you advice on details of the technique.