Automation of Solid-Phase Extraction for Urinary Opiate Analysis

The identification and confirmation of opiates extracted from biological matrices such as blood, plasma/serum, and urine may often lead to more questions than answers, as many exponents of forensic toxicology can attest to. The presence of morphine in samples of human urine may be an indicator of pain therapy medication, heroin abuse, or ingestion of foods containing poppy seeds such as bagels. Several studies have proposed the use of 6-acetylmorphine (also known as 6-MAM) as the primary biomarker for heroin abuse.1,2 Heroin (diacetylmorphine) is metabolized to 6-acetylmorphine within minutes of entering the human system; this is then conjugated or is further metabolized to morphine.1–4 Metabolism of heroin is shown in Figure 1.

Figure 1 - Heroin metabolism.

Thebaine is used as a biomarker for the ingestion of poppy seeds. These poppy seeds may contain varying concentrations of morphine, codeine, thebaine, and other opiate-type compounds. Thebaine undergoes significant degradation with exposure to heat, especially under acidic conditions such as those employed in derivatization procedures with N,O-bis(trimethylsilyl)- trifluoroacetamide (BSTFA)5 (a reagent found in many analytical laboratories). Laboratories at United Chemical Technologies, Inc. (UCT, Bristol, PA) have developed a method to detect and quantify 6-acetylmorphine, thebaine, and 10 other opiates in human urine using automated solid-phase extraction. The procedure involves enzymic hydrolysis (to deconjugate or free up the opiates) followed by solid-phase extraction (SPE) with a RapidTrace® SPE workstation (Caliper Life Sciences , Hopkinton, MA). Derivatization of the extracted opiates was followed by GC-MS in selective ion monitoring mode.

Materials and equipment

Acetic acid (glacial), ammonium hydroxide, ethyl acetate isopropranol, methanol, sodium acetate, and sodium phosphate (monobasic) were obtained from Fisher Scientific (Pittsburgh, PA). Propionic anhydride and β-glucuronidase from limpets were from Sigma-Aldrich (St. Louis, MO). Pyridine was attained from Regis Technologies (Morton Grove, IL). Analytical standards (6-acetylmorphine, codeine, hydrocodone, hydromorphone, morphine, morphine-d3 nalorphine norcodeine, normorphine, and oxycodone were obtained as methanolic solutions (1 mg/mL) from Cerilliant Corp. (Round Rock, TX). Naloxone and thebaine were acquired as powders from Sigma-Aldrich. Acetic acid was prepared as a 0.1-M solution with distilled water. Sodium acetate was prepared as a 0.1-M solution and adjusted to pH 4.5. Sodium phosphate was prepared as a 0.1-M solution with distilled water and adjusted to pH 6.

Table 1 - Analyte names, ions, and observed retention times for propyl derivatives

Solid-phase extraction was performed with a RapidTrace workstation using Clean Screen™ CSDAU SPE columns (200 mg sorbent mass in 3-mL tubes) from UCTGas chromatographic analysis of the extracts was performed on an GC coupled to a 5973A mass spectrometer (Agilent Technologies, Wilmington, DE) using an Rtx-5 capillary column (30 m × 0.25 mm i.d. with 0.25 μm film thickness) from Restek Corp. (Bellefonte, PA). The temperature program of the gas chromatograph was 100 °C (hold time 1 min) to 250 °C at 25 °C/min (hold time 2 min), then to 290 °C at 10 °C/min (hold time 0.5 min) and to 325 °C at 25 °C/min and held for 3.1 min. A sample injection volume of 1 μL was used. The monitored selected ions of the individual opiates and the respective retention times are shown in Table 1.

Sample pretreatment

Into screw-top culture tubes (13 x 100 mm) were added a solution of internal standard (morphine-d3 , 100 ng) and 4 mL of urine. This was followed by phosphate buffer (3 mL, pH 6) and 12,500 Fishman units of β-glucuronidase solution. Opiate addition was performed by introducing working solutions containing 12.5, 25, 50, 100, 250, 500, and 1000 ng of the individual opiates to each sample tube. The samples were mixed and hydrolyzed at 60 °C for 3 hr. The samples were cooled and loaded onto the automated SPE unit.

Automated SPE

Table 2 - RapidTrace workstation conditions

The reagents for solid-phase extraction consisted of methanol, distilled water, sodium acetate, and sodium phosphate buffers (0.1 M). A fresh solution of ethyl acetate:isopropanol:ammonium hydroxide (84:12:1) was used as the elution solvent. Conditions for the RapidTrace workstation are shown in Table 2. The lines of the workstation were purged with 10 mL of the appropriate solvent prior to use.

In essence, each solid-phase column was conditioned with methanol, distilled water, and phosphate buffer (0.1 M, pH 6). The samples were loaded onto the columns and allowed to pass through the sorbent, after which the columns were washed, dried, and eluted. The eluents were collected in their own separate tubes (12 × 75 mm).

Process after automated SPE

Figure 2 - Chromatogram of 25 ng extracted standard from spiked human urine.

Following sample elution, the liquids were transferred to borosilicate glass tubes (13 × 100 mm) and evaporated to dryness at 40 °C under a gentle stream of compressed air. To the sample in each tube was added 200 μL of a freshly made pyridine:propionic anhydride solution (1:1). The tubes were then capped and heated for 1 hr at 40 °C. The tubes were removed from the heat and evaporated to dryness at 40 °C. The residues were dissolved in 50 μL of a solution of ethyl acetate:methanol (70:30) and transferred to an autosampler vial for analysis by GC-MS. A chromatogram showing 25 ng of the different opiates extracted from urine is shown in Figure 2.