One form of Fentanyl therapy is the use of skin patches.
Over the past few years, it has been found that Fentanyl abusers will chew used skin patches – which can still contain 70% of the Fentanyl — that they pick up from trash bins.
It is also common for the user to not claim using the skin patch if they go to a withdrawal station. Fentanyl abuse in such a way can lead to problematic and dangerous withdrawal symptoms. Not knowing that Fentanyl was abused prevents proper therapy.
Currently, there are two immunological ways to detect Fentanyl: One is a urine stick that goes down to 10 ng/ml, and the other is a polyclonal antibody test that detects down to 1 ng/ml. Both tests only respond to Fentanyl. However, only 30% of the Fentanyl is excreted out of the metabolism in urine. The majority is excreted in urine as Norfentanyl.
Since today’s immunological tests can’t provide the right information, it is necessary to look into chromatographic methods that allow the detection of Fentanyl and Norfentanyl down to 0.1 ng/ml in urine.
From Sample to Result
Since the LOQ and LOD needed are very low (0.1 ng/ml) it is necessary to concentrate and clean up the samples by Solid Phase Extraction (SPE). This allows a concentration factor of up to 30 times, depending on the amount of sample used for extraction.
Afterward, the sample is introduced to an uHPLC system, and separation is performed on an analytical HPLC column. For the detection of the analytes, a high-resolution LC Orbitrap system is used to detect the exact mass and to gain sensitivity.
With the introduction of SOLA 30 mg to the market, there is new material available that allows high loadability, high ionexchange capacity and extremely clear extracts. SOLA is an organic polymer, such as Divinyl-Benzyl-Styrene (DVB), functioned with a ligand and combined with PTFE to remove frits. Its macro-porous design eliminates issues observed with traditional loose-packed SPE formats by combining the support material and active media components into a solid uniform sorbent bed.
The advantage of a polymer is the low pH in which it can be used. For the determination of Fentanyl and Norfentanyl, different SPE cleanup steps were tested.
It is necessary to modify the washing steps in order to get a clean extract. In case the specimen contains a lot of other medication, ion suppression can occur. Since every human matrix is different, over 100 urine samples were tested for ion suppression. The urine samples were spiked with 1 ng/ml Fentanyl, D5 and Norfentanyl. In general, urines with a lot of other medication (around 5% of all measured samples) showed ion suppression.
The other parameter that was tested, was the influence of the loading pH (pH in sample and on sorbent). Two different loading steps were tested since Fentanyl has a pKa of 8.3 and Norfentanyl of 10.3. At a loading pH of 6 (normally 2 units below pKa), both analytes were retained due to the hydrophobicity of the sorbent. However, at a pH of 6 the washing steps are limited, plus Norfentanyl detection limits were not low enough. Therefore, the big advantage of the polymer being used allowed loading conditions at a pH of 2 and used the full ability of the cation exchanger. That pH showed the best LODs and LOQs.
The final SPE method is shown in this figure:
One final point to optimize is the glass vials. The Pharmacopeia describes the first hydrolytic class in chapter 3.2. To gain full sensitivity for the detection of Fentanyl and Norfentanyl, it’s important to choose the right glass quality. Chromacol GOLD vials have only 29 % of free silanol groups on the surface. This makes the ideal vials for the analysis of compounds containing steric hindered nitrogen, halogenated substances and sulfur or phosphor-containing species. If analytes stick to the surface of the glass, they can’t be injected into the HPLC and the detection limits will decrease.
Since there is a necessity to detect Fentanyl and Norfentanyl after the abuse (chewing) of skin patches, a method development was performed on a high-resolution LC Orbitrap. The cleanest results could be accomplished with SOLA 30 mg SCX cartridges. This material is so outstanding in its characteristics, that it can even be used on a GCMS. It allows the lab to use it, not only for Fentanyl analysis but also for therapeutical drug monitoring on LCMS, as well as general unknown screening for drugs-of-abuse on GCMS.
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Ion suppression is an issue even using exact mass technology. By choosing the right HPLC column selectivity, the analytes of interest can be moved outside the ion suppression front. Due to its high carbon load and given the fact that the Acclaim C30 column is a spherical silica material with a 200 Angstrom pore size, it performed the best for the analysis of Fentanyl and Norfentanyl, moving both analytes outside the ion suppression. Together with highly inert glass vials, it was possible to achieve and LOD of 0.5 ng/ml and an LOQ of 0.1 ng/ml. Both limits a factor 10 below the actual detection limits of immunological tests.
 Disposition of toxic drugs and chemicals in Man, eleventh edition, Randall C. Baselt, Biomedical Publications, Seal Beach, California, pages 883-886
 EU Pharmacopeia chapter 3.2