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Technical Briefs |
1
Department of Clinical Laboratory Sciences, Louisiana State University Health Sciences Center, New Orleans, LA 70112-2262
2
LabOne, Inc., Lenexa, KS 66219-9752
3
LabCorp, Memphis, TN 38118
a author for correspondence: fax 504-568-6761,
lbrous{at}lsuhsc.edu
Several authors have reviewed existing methods (1)(2)(3)(4)(5)(6)(7)(8)(9) or presented new techniques (6)(7)(8)(9)(10)(11) for the analysis and separation of codeine, morphine, and the keto-opiates hydrocodone, hydromorphone, oxycodone, and oxymorphone. We present a modification of previously published procedures (6)(10) that incorporates the use of methoxyamine after enzymatic hydrolysis to form methoxime derivatives of the keto-opiates, which are then extracted using solid-phase columns and derivatized with propionic anhydride/pyridine.
We used a gas chromatography–mass spectrometry system composed of a model 5890 gas chromatograph with splitless injection, a model 5970 mass-selective detector (both from Hewlett Packard), and a DB-5 capillary column [15 m x 0.25 mm (i.d.); 0.25 µm film thickness; J&W Scientific]; helium (flow rate, 0.7 mL/min; linear velocity, 38 cm/s) was used as the carrier gas. The temperature program was as follows: initial temperature, 185 °C; ramp at 25 °C/min to 240 °C; hold for 0.5 min; ramp at 5 °C/min to 250 °C, then 40 °C/min to 290 °C; hold for 1.0 min. The injection temperature was 260 °C, and the transfer line temperature was 290 °C.
The following were obtained from Radian Corporation: (a) codeine, morphine, hydrocodone, hydromorphone, and oxycodone, which were used to prepare calibrators; (b) deuterated codeine, morphine, hydrocodone, and hydromorphone, which were used as internal standards; and (c) oxymorphone and norcodeine, which were used for interference studies. Methoxyamine hydrochloride, 2,4-dimethylaminopyridine, and propionic anhydride were obtained from Sigma. All other solvents and reagents were of reagent- or HPLC-grade quality.
A 2.0-mL urine sample was combined with 100 µL of a 6 mg/L internal standard solution and 1.0 mL of a 0.1 mol/L acetate buffer (pH 4.0) in an appropriately labeled 16 x 100 mm screw-cap tube. After mixing, conjugates were hydrolyzed by the addition of 150 µL of ß-glucuronidase solution (99.2 U/L; type L-II Patella vulgata; Sigma) to all tubes and incubation at 60 °C for 2 h. After hydrolysis, the keto-opiates were derivatized by the addition of 350 µL of 6 mol/L HCl and 250 µL of 100 g/L methoxyamine hydrochloride to each tube, followed by vortex mixing and incubation for 15 min at room temperature. Two milliliters of 1.5 mol/L phosphate buffer (pH 7.0) and 200 µL of 10 mol/L NaOH were added to each tube. After mixing and centrifugation, each supernatant was placed on a Bond Elute CertifyTM extraction column that had been activated by the sequential addition and elution of 2 mL of methanol and 2 mL of deionized water. Columns were then washed by sequential addition and elution of 2 mL of water, 2 mL of 0.1 mol/L acetate buffer (pH 4.0), and 6 mL of methanol. After the columns were dried under reduced pressure, analytes were eluted with 2 mL of freshly prepared dichloromethane:isopropyl alcohol:ammonium hydroxide (79:19:2 by volume). The eluates were dried under nitrogen at 40 °C, the extracted residues were reconstituted with 100 µL of 1 g/L propionic anhydride in pyridine, and the capped tubes were heated for 20 min at 70 °C. After cooling, 200 µL of methanol was added to each tube, followed by evaporation to dryness under nitrogen at 40 °C. The residue was reconstituted with 300 µL of ethyl acetate and transferred to a correspondingly labeled autoinjector vial; 1 µL was then injected into the system with a HP 6890 Automatic Liquid Sampler (Hewlett Packard).
Using the selected-ion monitoring mode and a HP DOS ChemstationTM data system (Hewlett Packard), we monitored the following ions (quantitative ions in parentheses) for the derivatized analytes: codeine, (m/z 355), 282, and 356; codeine-d3, (m/z 358) and 359; morphine (m/z 341), 397, and 268; morphine-d3, (m/z 344) and 400; hydrocodone, (m/z 328), 297, and 329; hydrocodone-d3, (m/z 331) and 332; hydromorphone, (m/z 314), 370, and 283; hydromorphone-d3 (m/z 317) and 373; oxycodone (m/z 400), 343, and 230. Quantification was based on an extracted calibrator (300 µg/L; ElSohly Laboratories, Inc.) for all five opiates. For expediency and economy, hydromorphone-d3 was used as the internal standard for the quantification of oxycodone as well as hydromorphone. Results using this common internal standard are acceptable, but use of oxycodone-d6 would be preferable.
The total-ion chromatographs of the propionyl derivatives of codeine
and morphine, the propionyl-oxime derivatives of hydromorphone and
oxycodone, and the methoxime derivative of hydrocodone are shown in
Fig. 1
. The chromatographic peaks are gaussian-shaped and demonstrate
near-baseline resolution with all peaks eluted within 6.5 min. This
resolution remains for the life of the column, which is 
2000
injections.
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Criteria for linearity included quantification within 20% of target concentration (12 calibrators; 60–10 000 µg/L for each opiate) and acceptable ion ratios (± 20%). The upper limit of linearity was 5000 µg/L for codeine, 8000 µg/L for morphine, 6000 µg/L for hydrocodone, and 3000 µg/L for hydromorphone and oxycodone.
Between-run precision (n = 31–52) around the 300 µg/L cutoff was determined monthly using data obtained from daily analysis of samples with target concentrations of 240 and 360 µg/L. Representative (most recent) CVs at these concentrations were as follows: 3.8% and 3.1% for codeine, 3.9% and 3.2% for morphine, 3.3% and 3.6% for hydrocodone, 3.2% and 3.9% for hydromorphone, and 4.4% and 4.5% for oxycodone, respectively.
Possible cross-interference with the quantification of codeine, morphine, hydrocodone, hydromorphone, and oxycodone was assessed by supplementing urine samples with 5000 µg/L oxymorphone and norcodeine (the two most likely interfering substances) in the presence of codeine, morphine, hydrocodone, hydromorphone, and oxycodone at 300 and 120 µg/L. In all cases, quantification of the analyte of interest was not affected by the presence of the potential interfering substance. Additionally, no cross-interference between the five opiates of interest at concentrations up to 10 000 µg/L was observed. Large concentrations of oxymorphone can interfere with the m/z 268 qualifier ion of morphine, but this does not interfere with the quantification of morphine.
The limit of detection (LOD) and limit of quantification (LOQ) determined by duplicate analysis of serially diluted samples were equal for each of the five analytes. The LODs and LOQs were 60 µg/L for codeine, hydrocodone, and hydromorphone; 90 µg/L for oxycodone; and 120 µg/L for morphine. The criteria for both LOD and LOQ included acceptable chromatography and acceptable ion ratios. Quantification within 20% of the target concentration was required for the LOQ but not the LOD.
In conclusion, we present a method that allows the simultaneous
quantification of codeine, morphine, hydrocodone, hydromorphone, and
oxycodone at concentrations from a minimum of 60–120 µg/L (LOQ for
each opiate) to a maximum of 5000 µg/L for codeine, 8000 µg/L for
morphine, 6000 µg/L for hydrocodone, and 3000 µg/L for
hydromorphone and oxycodone. The method demonstrates acceptable
precision and lack of cross-interference and interference from other
opiates, uses a relatively small sample volume (2.0 mL), and has an
analysis time of 6.5 min. This method has been used in the
laboratory (Memphis, TN) for the analysis of >3500 samples in a
4-month period and has been found to be reliable as demonstrated by
calibrator and control reproducibility and the absence of interference.
References
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