To the Editor:
Our laboratory offers a multianalyte LC-MS/MS urine drug testing panel to assist with compliance monitoring for patients enrolled in chronic pain management programs (1). Recently, we investigated switching from a plastic-only urine collection container (Globe Scientific; item number: 6220) to a metal lid type (Covidien; item number: 2200SA). Both of the containers, not including the lids, were composed of polypropylene. Low-density polyethylene (LDPE)1 lined the inside of the metal lid to ensure a secure fit when closed. The plastic-only container did not have a liner. The quantitative influence of the specimen container was originally investigated by splitting spiked urine specimens and inverting the metal lid container to ensure contact with the lid. For all of the analytes on the panel, no difference >20% was observed except for Δ9-tetrahydrocannabinol (THC)-COOH, for which levels from the metal lid containers were significantly lower than their upright plastic-only counterparts. Other positions were compared to the upright plastic-only containers, such as inverted plastic only and upright metal lid. The THC-COOH percent differences vs the upright plastic-only containers (+4% to −12%) were similar to the CVs found during the method's original validation for interday and intraday precision (4.6%–9.8%).
Herein is the further investigation of the inverted metal lid containers on THC-COOH quantification with positive THC-COOH patient samples and spiked THC-COOH and THC-COOH glucuronide (THC-COOH-Glu) urine. Both THC-COOH and THC-COOH-Glu were investigated although THC-COOH-Glu is the major urinary metabolite (2). Our method quantified THC-COOH (the precursor to THC-COOH-Glu) and THC-COOH-Glu together because of specimen enzymatic hydrolysis before LC-MS/MS analysis (1). However, the effect on the individual components could be measured in the spiked urines because only 1 component was added to THC-COOH–free urine. Use of leftover patient specimens was approved by the Cleveland Clinic Institutional Review Board.
First, THC-COOH or THC-COOH-Glu was spiked into a THC-COOH–free patient urine pool at the following concentrations: 2000, 750, and 50 ng/mL (high, medium, and low). A total of 10 mL of each specimen was placed into the upright plastic-only or inverted metal lid container and stored at 4 °C. After 24 h, each specimen was removed from the container and stored at −20 °C in a polystyrene test tube until analysis. The percent difference from the upright plastic-only container was then calculated. The THC-COOH levels were found to be lower in the inverted, metal lid containers for 5 out of the 6 experiments using different THC-COOH and THC-COOH-Glu concentrations (Fig. 1A). The one exception, the sample named low THC-COOH, had little difference between the plastic-only and metal lid containers: 31 vs 34 ng/mL, respectively. For the other specimens, THC-COOH concentrations fell by over 20% in the metal lid containers compared to the plastic-only ones.
The experiment was then repeated with THC-COOH–positive leftover patient specimens in place of spiked THC-COOH or THC-COOH-Glu urine. In 4 out of the 5 patients, the THC-COOH levels were lower in the inverted, metal lid containers (Fig. 1B). The exception was patient specimen 4, which displayed a small difference between the upright plastic-only and inverted metal lid containers: 55 vs 60 ng/mL, respectively. For the other patient specimens, there was over a 20% reduction in THC-COOH concentration in the inverted metal lid containers compared to the upright plastic-only ones.
We hypothesized that the LDPE lining was responsible for lowering the THC-COOH levels. This hypothesis was tested by removing the LDPE lining from the metal lid and placing it into a plastic-only container. The 10 mL spiked THC-COOH or THC-COOH-Glu urine was then added to the upright containers. The removed LDPE was completely submerged with urine in the upright containers. The specimens were stored at 4 °C for 24 h. After 24 h, each urine specimen was removed and stored at −20 °C in a polystyrene test tube until analysis. The percent differences from the upright plastic-only container control were then calculated. All of the THC-COOH levels were lower for the LDPE-added containers than the plastic-only controls (Fig. 1C). With the extreme reduction in THC-COOH levels (77% to 87%, not including the <16 ng/mL samples), this experiment indicates that the LDPE lining caused the decreased urine THC-COOH concentrations. Urine THC-COOH losses at 4 °C have been previously reported with high-density polyethylene (up to approximately 17%) and polypropylene containers (up to approximately 14%) (3). To our knowledge, this is the first systemic investigation on THC-COOH losses in urine from contact with LDPE.
Because of limitations of available leftover patient urine volume, analysis time, and cost, we only performed singlicate experiments, which demonstrated that the THC-COOH concentrations in the inverted, metal lid containers of urine specimens varied by −50% to +9% from their plastic-only counterparts. When averaged, a reduction of 29% was observed. Specimens collected and stored in these containers could potentially yield falsely low THC-COOH results. We caution the adoption of new specimen containers until they are investigated on all subjected analytes.
↵1 Nonstandard abbreviations:
- low-density polyethylene
Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest.
- Received October 17, 2016.
- Accepted November 21, 2016.
- © 2016 American Association for Clinical Chemistry